Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

Apparent translocation of glucose transport activity in rat epididymal adipocytes by insulin-like effects of high pH or hyperosmolarity

The basal and plus insulin states of glucose transport activity in adipocytes are known to show different responses to changes in the pH or osmolarity of the incubation mixture. When the pH was raised from 7 to 8, the basal glucose transport activity (assessed from the rate of 3-O-methyl-D-glucose uptake) was increased approximately 3-fold while the plus insulin activity remained virtually unaffected. Likewise, when cells were exposed to 300 mM sorbitol, the basal glucose transport activity, but not the plus insulin activity, was considerably increased. In both cases, the change in the transport activity was ATP-dependent and was completed in approximately 60 min. The increase in the cellular glucose transport activity was accompanied, in both cases, by an increase in the glucose transport activity in the plasma membrane fraction and a decrease in the activity in the high-speed pellet fraction. The transport activity in the subcellular fractions was determined after reconstitution into egg lecithin liposomes. Both isotonic buffer at pH 8.0 and hypertonic buffer at pH 7.4 significantly stimulated membrane-bound cAMP phosphodiesterase in adipocytes. It is concluded that the above two experimental conditions may induce insulin-like effects in fat cells and may facilitate translocation of the glucose transport activity from an intracellular site to the plasma membrane.     

Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation.

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes

To study molecular mechanisms for glucosamine-induced insulin resistance, we induced complete and reversible insulin resistance in 3T3-L1 adipocytes with glucosamine in a dose- and time-dependent manner (maximal effects at 50 mM glucosamine after 6 h). In these cells, glucosamine impaired insulin-stimulated GLUT-4 translocation. Glucosamine (6 h) did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 and -2 and weakly, if at all, impaired insulin stimulation of phosphatidylinositol 3-kinase. Glucosamine, however, severely impaired insulin stimulation of Akt. Inhibition of insulin-stimulated glucose transport was correlated with that of Akt activity. In these cells, glucosamine also inhibited insulin stimulation of p70 S6 kinase. Glucosamine did not alter basal glucose transport and insulin stimulation of GLUT-1 translocation and mitogen-activated protein kinase. In summary, glucosamine induced complete and reversible insulin resistance in 3T3-L1 adipocytes. This insulin resistance was accompanied by impaired insulin stimulation of GLUT-4 translocation and Akt activity, without significant impairment of upstream molecules in insulin-signaling pathway.     

Phenylarsine oxide and the mechanism of insulin-stimulated sugar transport

The actions of phenylarsine oxide (PAO) on hormone receptors and transport processes are reviewed with particular reference to the mechanism of insulin-stimulated sugar transport. It is suggested that as well as reaction with vicinal -SH groups, vicinal -SH/-OH and -SH/-CO2H groups should also be considered as potential reaction sites for PAO. The relatively high levels of these vicinal combinations of groups in many hormone receptors makes them particularly susceptible to reaction with PAO. In the case of insulin-stimulated sugar transport PAO does not inhibit insulin binding to its receptor at low concentrations but may react directly with the glucose transporters in some cells. A hypothesis is proposed suggesting that PAO may react specifically with one transporter isoform (GLUT-4) which is found almost exclusively in rat adipocytes, skeletal muscle and heart tissue (i.e. insulin responsive tissue) whereas in insulin unresponsive cells such as erythrocytes the GLUT-1 isoform is the predominant transporter which is not inhibited by PAO.     

Effects of cellular ATP depletion on glucose transport and insulin signaling in 3T3-L1 adipocytes

Glucosamine induced insulin resistance in 3T3-L1 adipocytes, which was associated with a 15% decrease in cellular ATP content. To study the role of ATP depletion in insulin resistance, we employed sodium azide (NaN3) and dinitrophenol (DNP), which affect mitochondrial oxidative phosphorylation, to achieve a similar 15% ATP depletion. Unlike glucosamine, NaN3 and DNP markedly increased basal glucose transport, and the increased basal glucose transport was associated with increased GLUT-1 content in the plasma membrane without changes in total GLUT-1 content. These agents, like glucosamine, did not affect the early insulin signaling that is implicated in insulin stimulation of glucose transport. In cells with a severe 40% ATP depletion, basal glucose transport was similarly elevated, and insulin-stimulated glucose transport was similar in cells with 15% ATP depletion. In these cells, however, early insulin signaling was severely diminished. These data suggest that cellular ATP depletion by glucosamine, NaN3, and DNP exerts differential effects on basal and insulin-stimulated glucose transport and that ATP depletion per se does not induce insulin resistance in 3T3-L1 adipocytes.     

Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6 muscle cells in culture

The regulation by glucose and insulin of the muscle-specific facilitative glucose transport system GLUT-4 was investigated in L6 muscle cells in culture. Hexose transport activity, mRNA expression, and the subcellular localization of the GLUT-4 protein were analyzed. As observed previously (Walker, P. S., Ramlal, T., Sarabia, V., Koivisto, U.-M., Bilan, P. J., Pessin, J. E., and Klip, A. (1990) J. Biol. Chem. 265, 1516-1523), 24 h of glucose starvation and 24 h of insulin treatment each increase glucose transport activity severalfold. Here we report a differential regulation of the GLUT-4 and GLUT-1 transport systems under these conditions. (a) The level of GLUT-4 mRNA was not affected by glucose starvation and was diminished by prolonged (24 h) administration of insulin; in contrast, the level of GLUT-1 mRNA was elevated under both conditions. (b) Glucose starvation and prolonged insulin administration increased the amount of both GLUT-4 and GLUT-1 proteins in the plasma membrane. (c) In intracellular membranes, glucose starvation elevated, and prolonged insulin administration reduced, the GLUT-4 protein content. In contrast, the GLUT-1 protein content in these membranes decreased with glucose starvation and increased with insulin treatment. Glucose transport was rapidly curbed upon refeeding glucose to glucose-starved cells, with half-maximal reversal after 30 min and maximal reversal after 4 h. This was followed by a marked decrease in the levels of GLUT-1 mRNA without major changes in GLUT-4 mRNA. Neither 2-deoxy-D-glucose nor 3-O-methyl-D-glucose could substitute for D-glucose in these effects. It is proposed that glucose and insulin differentially regulate the two glucose transport systems in L6 muscle cells and that the rapid down-regulation of hexose transport activity by glucose is regulated by post-translational mechanisms.     

The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin- sensitive cells

Insulin stimulates glucose transport in adipocytes by translocation of the glucose transporter (GLUT-4) from an intracellular site to the cell surface. We have characterized different synaptobrevin/vesicle- associated membrane protein (VAMP) homologues in adipocytes and studied their intracellular distribution with respect to GLUT-4. VAMP-1, VAMP- 2, and cellubrevin cDNAs were isolated from a 3T3-L1 adipocyte expression library. VAMP-2 and cellubrevin were: (a) the most abundant isoforms in adipocytes, (b) detectable in all insulin responsive tissues, (c) translocated to the cell surface in response to insulin, and (d) found in immunoadsorbed GLUT-4 vesicles. To further define their intracellular distribution, 3T3-L1 adipocytes were incubated with a transferrin/HRP conjugate (Tf/HRP) and endosomes ablated following addition of DAB and H2O2. While this resulted in ablation of > 90% of the transferrin receptor (TfR) and cellubrevin found in intracellular membranes, 60% of GLUT-4 and 90% of VAMP-2 was not ablated. Immuno-EM on intracellular vesicles from adipocytes revealed that VAMP-2 was colocalized with GLUT-4, whereas only partial colocalization was observed between GLUT-4 and cellubrevin. These studies show that two different v-SNAREs, cellubrevin and VAMP-2, are partially segregated in different intracellular compartments in adipocytes, implying that they may define separate classes of secretory vesicles in these cells. We conclude that a proportion of GLUT-4 is found in recycling endosomes in nonstimulated adipocytes together with cellubrevin and the transferrin receptor. In addition, GLUT-4 and VAMP-2 are selectively enriched in a postendocytic compartment. Further study is required to elucidate the function of this latter compartment in insulin-responsive cells.     

Translocation of Insulin-Regulated Glucose Transporter Is Stimulated by Long-Chain 1,2-Diacylglycerol in Rat Adipocytes

The long-chain diacylglycerol 1,2-dimyristoylglycerol emulsified with taurodeoxycholate has been shown to potently stimulate glucose transport in isolated rat adipocytes (Strålfors, Nature 335, 554-556 (1988)). We now report that this 1,2-diacylglycerol in the presence of taurodeoxycholate, similarly to insulin, induced translocation of the insulin-regulated glucose transporter (GLUT-4) from a microsomal membrane compartment to the plasma membrane. H4IIE hepatoma cells expressed mRNA for GLUT-1, but not for GLUT-4. In these, otherwise insulin-responsive, cells diacylglycerol or insulin had only a marginal effect on glucose transport.     

Insulin stimulation of glucose transport in isolated rat adipocytes. Functional evidence for insulin activation of intrinsic transporter activity within the plasma membrane.

We examined the effects of the membrane-impermeant amino-group-modifying agent fluorescein isothiocyanate (FITC) on the basal and insulin-stimulated hexose-transport activity of isolated rat adipocytes. Pre-treatment of cells with FITC causes irreversible inhibition of transport measured in subsequently washed cells. Transport activity was inhibited by approx. 50% with 2 mM-FITC in 8 min. The cells respond to insulin, after FITC treatment and removal, and the fold increase in transport above the basal value caused by maximal concentrations of insulin was independent of the concentration of FITC used for pre-treatment over the range 0-2 mM, where basal activity was progressively inhibited. The ability of FITC to modify selectively hexose transporters accessible only to the external milieu was evaluated by two methods. (1) Free intracellular FITC, and the distribution of FITC bound to cellular components, were assessed after dialysis of the homogenate and subcellular fractionation on sucrose gradients by direct spectroscopic measurement of fluorescein. Most (98%) of the FITC was associated with the non-diffusible fractions. Equilibrium sucrose-density-gradient centrifugation of the homogenate demonstrated that the subcellular distribution of the bound FITC correlated with the density distribution of a plasma-membrane marker, but not markers for Golgi, endoplasmic reticulum, mitochondria or protein. Exposing the cellular homogenate, rather than the intact cell preparation, to 2 mM-FITC resulted in a 4-5-fold increase in total bound FITC, and the density-distribution profile more closely resembled the distribution of total protein. (2) Incubation of hexokinase preparations with FITC rapidly and irreversibly inactivates this protein. However, both intracellular hexokinase total activity and its apparent Michaelis constant for glucose were unaffected in FITC-treated intact cells. Further control experiments demonstrated that FITC pre-treatment of cells had no effect on the intracellular ATP concentration or the dose-response curve of insulin stimulation of hexose transport. Since the fold increase of hexose transport induced by insulin is constant over the range of inhibition of surface-labelled hexose transporters, we suggest that insulin-induced insertion of additional transporters into the plasma membrane may not be the major locus of acceleration of hexose transport by the hormone.     

Transport function and subcellular distribution of purified human erythrocyte glucose transporter reconstituted into rat adipocytes.

In order to delineate the insulin-independent (constitutive) and insulin-dependent regulations of the plasma membrane glucose transporter concentrations in rat adipocytes, we introduced purified human erythrocyte GLUT-1 (HEGT) into rat adipocytes by poly(ethylene glycol)-induced vesicle-cell fusion and its transport function and subcellular distribution in the host cell were measured. HEGT in adipocytes catalysed 3-O-methylglucose equilibrium exchange with a turnover number that is indistinguishable from that of the basal adipocyte transporters. However, insulin did not stimulate significantly the HEGT function in adipocytes where it stimulated the native transporter function by 7-8-fold. The steady state distribution and the transmembrane orientation assays revealed that more than 85% of the HEGT that were inserted in the physiological, cytoplasmic side-in orientation at the adipocytes plasma membrane were moved into low-density microsomes (LDM), while 90% of the HEGT that were inserted in the wrong, cytoplasmic side-out orientation were retained in the plasma membrane. Furthermore, more than 70% of the LDM-associated HEGT were found in a small subset of LDM that also contained 80% of the LDM-associated GLUT-4, the insulin-regulatable, native adipocyte glucose transporter. However, insulin did not cause redistribution of HEGT from LDM to the plasma membrane under the condition where it recruited GLUT-4 from LDM to increase the plasma membrane GLUT-4 content 4-5-fold. These results demonstrate that the erythrocyte GLUT-1 introduced in adipocytes transports glucose with an intrinsic activity similar to that of the adipocyte GLUT-1 and/or GLUT-4, and enters the constitutive GLUT-4 translocation pathway of the host cell provided it is in physiological transmembrane orientation, but fails to enter the insulin-dependent GLUT-4 recruitment pathway. We suggested that the adipocyte plasma membrane glucose transporter concentration is constitutively kept low by a mechanism where a cell-specific constituent interacts with a cytoplasmic domain common to GLUT-1 and GLUT-4, while the insulin-dependent recruitment requires a cytoplasmic domain specific to GLUT-4.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

IIb group metal ions (Zn2+, Cd2+, Hg2+) stimulate glucose transport activity by post-insulin receptor kinase mechanism in rat adipocytes

Zn2+ (1 mM), Cd2+ (1 mM), and Hg2+ (0.1 mM) belonging to the IIb group in the periodic table stimulated glucose transport activity and cAMP phosphodiesterase in rat adipocytes. The stimulation of glucose transport was due to the translocation of glucose transporters from the intracellular site to the plasma membrane. However, in intact adipocytes none of these ions stimulated insulin receptor kinase activity or phosphorylation of the 95-kDa subunit of insulin receptor or 170- or 60-kDa proteins at the tyrosyl residues. These proteins were markedly phosphorylated by addition of 0.3 nM insulin which stimulated glucose transport activity as effectively as these metal ions. These results indicate that Zn2+, Cd2+, and Hg2+ mimic insulin action by a post-receptor/kinase mechanism.     

Impaired insulin-mediated inhibition of lipolysis and glucose transport with aging

Regulation of hormone action with aging has been extensively studied; adipocytes provide an interesting model for some of these questions. We have compared the ability of insulin to stimulate glucose uptake and suppress lipolysis in adipocytes isolated from two month and twelve month-old rats. The ability of insulin to stimulate maximal glucose transport was decreased in adipocytes from the older rats (P less than 0.001); as well, insulin's EC50 was also higher (P less than 0.01) in these cells. Furthermore, these defects were present when insulin-stimulated glucose transport was measured in the presence or absence of adenosine deaminase which metabolizes endogenously released adenosine. Endogenously released adenosine is a stimulator of glucose transport and an inhibitor of lipolysis. Maximal suppression of isoproterenol-induced lipolysis by insulin was similar when adipocytes isolated from the two age groups were incubated in the absence of adenosine deaminase. However, maximal insulin-mediated suppression of lipolysis was found to be significantly decreased (P less than 0.001) in adipocytes isolated from older rats when the experiments were done in the presence of adenosine deaminase; also, insulin's EC50 was increased in these cells under these conditions (P less than 0.001). These results emphasize the importance of the adenosine receptor in modulating the response of isolated adipocytes to insulin, particularly for lipolysis, and document the presence of age-associated defects in insulin regulation of both glucose transport and lipolysis.     

Transport function and subcellular distribution of purified human erythrocyte glucose transporter reconstituted into rat adipocytes

In order to delineate the insulin-independent (constitutive) and inssulin-dependent regulations of the plasma membrane glucose transporter concentrations in rat adipocytes, we introduced purified human erythrocyte GLUT-1 (HEGT) into rat adipocytes by poly(ethylene glycol)-induced vesicle-cell fusion and its transport function and subcellular distribution in the host cell were measured. HEGT in adipocytes catalysed 3-O-methylglucose equilibrium exchange with a turnover number that is indistinguishable from that of the basal adipocyte transporters. However, insulin did not stimulate significantly the HEGT function in adipocytes where it stimulated the native transporter function by 7-8-fold. The steady state distribution and the transmembrane orientation assays revealed that more than 85% of the HEGT that were inserted in the physiological, cytoplasmic side-in orientation at the adipocytes plasma membrane were moved into low-density microsomes (LDM), while 90% of the HEGT that were inserted in the wrong, cytoplasmic side-out orientation were retained in the plasma membrane. Furthermore, more than 70% of the LDM-associated HEGT were found in a small subset of LDM that also contained 80% of the LDM-associated GLUT-4, the insulin-regulatable, native adipocyte glucose transporter. However, insulin did not cause redistribution of HEGT from LDM to the plasma membrane under the condition where it recruited GLUT-4 from LDM to increase the plasma membrane GLUT-4 content 4–5-fold. These results demonstrate that the erythrocyte GLUT-1 introduced in adipocytes transports glucose with an intrinsic activity similar to that of the adipocyte GLUT-1 and/or GLUT-4, and enters the constitutive GLUT-4 translocation pathway of the host cell provided it is in physiological transmembrane orientation, but fails to enter the insulin-dependent GLUT-4 recruitment pathway. We suggested that the adipocyte plasma membrane glucose transporter concentration is constitutively kept low by a mechanism where a cell-specific constitutent interacts with a cytoplasmic domain common to GLUT-1 and GLUT-4, while the insulin-dependent recruitment requires a cytoplasmic domain specific to GLUT-4.     

The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.     

Rapamycin partially prevents insulin resistance induced by chronic insulin treatment

Chronic insulin exposure induces serine/threonine phosphorylation and degradation of IRS-1 through a rapamycin-sensitive pathway, which results in a down-regulation of insulin action. In this study, to investigate whether rapamycin (an mTOR inhibitor) could prevent insulin resistance induced by hyperinsulinemia, 3T3-L1 adipocytes were incubated chronically in the presence of insulin with or without the addition of rapamycin. Subsequently, the cells were washed and re-stimulated acutely with insulin. Chronic insulin stimulation caused a reduction of GLUT-4 and IRS-1 proteins with a correlated decrease in acute insulin-induced PKB and MAPK phosphorylations as well as a reduction in insulin-stimulated glucose transport. Rapamycin prevented the reduction of IRS-1 protein levels and insulin-induced PKB Ser-473 phosphorylation with a partial normalization of insulin-induced glucose transport. In contrast, rapamycin had no effect on the decrease in insulin-induced MAPK phosphorylation or GLUT-4 protein levels. These results suggest that chronic insulin exposure leads to a down-regulation of PKB and MAPK pathways through different mechanisms in adipocytes.     

Vanadate induces the recruitment of glut-4 glucose transporter to the plasma membrane of rat adipocytes

Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter.     

Diversity in the effects of extracellular ATP and adenosine on the cellular processing and physiologic actions of insulin in rat adipocytes

ATP or adenosine (1 mM) added to extracellular buffer abolished both chloroquine- and monensin-dependent accumulation of [125I]iodoinsulin in isolated rat adipocytes. The effects of ATP were not secondary to its conversion to adenosine and were mimicked by beta, gamma-methyleneadenosine 5'-triphosphate. ATP, but not adenosine, partially inhibited the binding of insulin to the cellular receptor. Neither ATP nor adenosine had any significant effect on both internalization of cell-bound insulin and externalization of the internalized hormone. The degradation of cell-bound insulin was reduced to a considerable extent by both 0.1 mM chloroquine and 5 mM ATP, to a lesser degree by 1 mM ATP, and not significantly by 1 or 5 mM adenosine. Physiologically, (a) 1 mM ATP had a strong, while 1 mM adenosine had a mild inhibitory effect on the insulin-stimulated glucose transport without affecting its basal activity, (b) both ATP and adenosine moderately stimulated basal as well as insulin-stimulated glycogen synthase, and (c) ATP, but not adenosine, transiently stimulated basal cAMP phosphodiesterase without affecting the insulin-stimulated enzyme. Phosphodiesterase in cells that had been exposed to ATP for 30 min was refractory to ATP added afresh, but not to insulin. These data suggest that (a) extracellular ATP may block the degradative pathway of insulin processing, (b) adenosine might render the ordinarily irreversible intracellular traffic of insulin reversible or modulate a pathway which is yet to be identified, (c) the previously reported effect of ATP on glycogen synthase may not involve phosphorylation, (d) ATP stimulates cAMP phosphodiesterase by a mechanism which is distinct from that of insulin, and (e) the degradative pathway of insulin processing may not be involved in the physiologic actions of the hormone on glycogen synthase and phosphodiesterase.